The cAMP-dependent protein kinase (PKA), the prime target of the second messenger cAMP in mammalian cells, is activated by the binding of cAMP to the regulatory subunit (R), of the molecule and results in the release of the active catalytic kinase subunit (C). There are several isoforms of both subunits of PKA. All members of the PKA family share significant sequence homology and perform signal transduction via protein phosphorylation. Almost all cell types express one or more isoforms of PKA. The protein inhibitor of PKA (PKI) acts by binding with high affinity to the substrate binding site of the free active catalytic subunit (Walsh D. A. (1990) Peptides and Protein Phosphorylation, CRC, Boca Raton, Fla., pp. 43-84). In addition, PKI has been shown to export the C subunit from the nucleus (Fantozzi D. A. et al (1994) J Biol Chem 269: 2676-2686).
The endogenous inhibitor of cAMP-dependent protein kinase (PKA) is down-regulated in the kidneys from vitamin-D-replete chicks as compared to vitamin-D-deficient chicks. Screening of a vitamin-D-deficient chick kidney library resulted in the isolation of a 450-bp cDNA clone encoding the 76-amino acid protein kinase inhibitor (Marchetto G. S. and Henry H. L. (1995) Gene 158: 303-304). The deduced amino acid sequence of avian PKI shares 80% and 41% identity with the mammalian PKI alpha and PKI beta 1 isoforms, respectively.
Both avian PKI and human PKI alpha share conserved N-terminal sequences, including the pseudo-substrate site (18GRRNA22), which are required for potent inhibition of the catalytic subunit of PKA (Marchetto and Henry, supra). The amino acid phenylalanine at position 11 plays a critical role in PKI-C subunit binding (Baude et al (1994) J Biol Chem 269: 18128-18133).
Cell cycle synchrony studies suggests that PKI has an important role in the inhibition of nuclear C subunit activity that is required for cell cycle progression (Wen W. et al (1995) J Biol Chem 270: 2041-2046). Microinjection of PKI alpha antibody prevented the cell cycle progression of serum-starved cells.